Use of liquid chromatography and mass spectrometry to characterize oligonucleotides

Abstract

The disclosure provides methods of characterizing a sample of oligonucleotides of interest using liquid chromatography and mass spectrometry.

What is claimed is: 
     
       1. A method of making a composition comprising oligonucleotides of interest with high purity, the method comprising:
 a. synthesizing a population of oligonucleotides of interest, thereby providing a sample comprising the population of oligonucleotides of interest and at least one impurity comprising at least one additional population of oligonucleotides, wherein individual oligonucleotides of interest in the population have identical sequences; 
 b. subjecting the sample to liquid chromatography and mass spectrometry, wherein the liquid chromatography comprises Ion-pairing Reversed-Phase Liquid Chromatography (IP-RPLC), and wherein the IP-RPLC comprises a mobile phase comprising a first buffer comprising 40-60 mM Hexafluoroisopropanol (HFIP) and 3-15 mM N,N-Diisopropylethylamine (DIEA) in water and a second buffer comprising 40-60 mM HFIP and 3-15 mM DIEA in acetonitrile, thereby generating at least one mass spectrogram corresponding to the population of oligonucleotides of interest and a mass spectrogram corresponding to the at least one additional population of oligonucleotides; 
 c. determining a percentage of total oligonucleotides in the sample corresponding to the population of oligonucleotides of interest; and 
 d. selecting a sample comprising a population of the oligonucleotides of interest with high purity for the composition, 
 thereby making the composition comprising oligonucleotides of interest with high purity. 
 
     
     
       2. The method of  claim 1 , wherein the additional population of oligonucleotides comprises a fragmentation product of or a synthesis byproduct of the oligonucleotides of interest. 
     
     
       3. The method of  claim 1 , further comprising determining the percentage of total oligonucleotides in the sample corresponding to the at least one additional population of oligonucleotides. 
     
     
       4. The method of  claim 1 , wherein the oligonucleotides of interest are deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or DNA-RNA hybrids. 
     
     
       5. The method of  claim 1 , wherein the oligonucleotides of interest are single stranded or double stranded. 
     
     
       6. The method of  claim 1 , wherein the oligonucleotides of interest comprise a hairpin or stem-loop structure. 
     
     
       7. The method of  claim 1 , wherein the oligonucleotides of interest are between 15 and 100 nucleotides in length. 
     
     
       8. The method of  claim 1 , wherein the oligonucleotides of interest are therapeutic oligonucleotides. 
     
     
       9. The method of  claim 8 , wherein the therapeutic oligonucleotides comprise antisense oligonucleotides (ASO), dsRNAs, siRNAs, aptamers or microRNAs. 
     
     
       10. The method of  claim 1 , wherein the oligonucleotides of interest comprise at least one modification. 
     
     
       11. The method of  claim 10 , wherein the at least one modification is at the 5′ end, the 3′ end, an internal nucleobase, or a combination thereof, of individual oligonucleotides of interest. 
     
     
       12. The method of  claim 10 , wherein the at least one modification comprises a locked nucleic acid (LNA), a phosphorothioate (PS) linkage, a terminal 5′ or 3′ phosphate (PO), a 5′ methyl (5-Me) modification, a 2′-O-Methyl (2′-O-Me) modification, a 2′-O-methoxyethyl (2′-MOE) modification, a constrained ethyl (cET) nucleoside analog, a polyethylene glycol (PEG) or a combination thereof. 
     
     
       13. The method of  claim 1 , wherein the mobile phase comprises a first buffer comprising 50 mM HFIP and 5 mM DIEA in water and a second buffer comprising 50 mM HFIP and 5 mM DIEA in acetonitrile. 
     
     
       14. The method of  claim 1 , wherein the IP-RPLC comprises a column with a mean nominal particle size of 1.7 μm, a median particle pore size of 130 Å, a column length 100 mm and a 2.1 mm inner diameter. 
     
     
       15. The method of  claim 1 , wherein the mass spectrometry comprises electrospray ionization (ESI). 
     
     
       16. The method of  claim 1 , wherein the mass spectrometry is tandem mass spectrometry (MS/MS). 
     
     
       17. The method of  claim 1 , wherein the mass spectrometry is tandem mass spectrometry (MS/MS) and wherein the MS/MS comprises fragmentation of the population of oligonucleotides of interest, the at least one additional population of oligonucleotides, or a combination thereof. 
     
     
       18. The method of  claim 17 , wherein the fragmentation comprises higher-energy collisional dissociation (HCD) comprising a normalized collisional energy (NCE) of 15% to 35%. 
     
     
       19. The method of  claim 1 , wherein step (c) comprises determining the intact mass of the oligonucleotides of interest. 
     
     
       20. The method of  claim 1 , wherein step (c) further comprises determining the intact mass of the at least one additional population of oligonucleotides. 
     
     
       21. The method of  claim 1 , wherein step (c) comprises determining the structure of the oligonucleotides of interest using mass spectrometry. 
     
     
       22. The method of  claim 1 , wherein step (c) further comprises determining the structure of the at least one additional population of oligonucleotides. 
     
     
       23. The method of  claim 1 , wherein at least 90% of the total oligonucleotides in the composition are the oligonucleotide of interest. 
     
     
       24. The method of  claim 1 , further comprising step (e) adding a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
       25. The method of  claim 1 , wherein step (d) further comprises discarding or further purifying the sample if the sample is not of high purity. 
     
     
       26. A method of making a composition comprising oligonucleotides of interest, wherein at least 90% of the oligonucleotides in the composition are the oligonucleotide of interest, the method comprising:
 a. synthesizing a population of oligonucleotides of interest, thereby providing a sample comprising the population of oligonucleotides of interest of identical sequence and/or modification, and at least one impurity comprising at least one additional population of oligonucleotides; 
 b. subjecting the sample to liquid chromatography and tandem mass spectrometry (MS/MS), 
 wherein the liquid chromatography comprises:
 i. hydrophilic interaction liquid chromatography (HILIC) comprising a mobile phase, wherein the mobile phase comprises a first buffer comprising 15 mM ammonium formate or ammonium acetate in 70% acetonitrile (ACN), and a second buffer comprising 15 mM ammonium formate or ammonium acetate in 30% ACN, or 
 ii. Ion-pairing Reversed-Phase Liquid Chromatography (IP-RPLC) comprising a mobile phase, wherein the mobile phase comprises a first buffer comprising 50 mM Hexafluoroisopropanol (HFIP) and 5 mM N,N-Diisopropylethylamine (DIEA) in water and a second buffer comprising 50 mM HFIP and 5 mM DIEA in acetonitrile; 
 
 wherein the MS/MS comprises fragmentation of the population of oligonucleotides of interest and the additional population of oligonucleotides in the sample using higher-energy collisional dissociation (HCD) comprising a normalized collisional energy (NCE) of 15% to 35%, 
 thereby generating at least one mass spectrogram corresponding to the population oligonucleotides of interest and a mass spectrogram corresponding to the additional population of oligonucleotides; 
 c. determining a percentage of total oligonucleotides in the sample corresponding to the population of oligonucleotides of interest; and 
 d. selecting a sample comprising a population of the oligonucleotides of interest, wherein at least 90% of the oligonucleotides in the composition are the oligonucleotide of interest, for the composition, 
 thereby making the composition comprising oligonucleotides of interest. 
 
     
     
       27. The method of  claim 26 , further comprising step (e) adding a pharmaceutically acceptable carrier, diluent or excipient. 
     
     
       28. The method of  claim 26 , wherein step (d) further comprises discarding or further purifying the sample if less than 90% of the oligonucleotides in the composition are the oligonucleotide of interest.